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Cellular Communication Kamil Růžička FGP CEITEC MU Genomics Lectures.

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Presentation on theme: "Cellular Communication Kamil Růžička FGP CEITEC MU Genomics Lectures."— Presentation transcript:

1 Cellular Communication Kamil Růžička FGP CEITEC MU Genomics Lectures

2 How does fluorescence work?

3 How does a fluorescence microscope work?

4 How does a confocal microscope work?

5 Protein localization live imaging Martin Chalfie GFP discovery - Nobel Prize 2008 Roger Tsien Osamu Shimomura

6 GFP fusions your genepromoter here can be GFP terminator your genepromoter terminator your genepromoter terminator here can be GFP N-terminal fusion C-terminal fusion fusion inside the coding sequence

7 GFP and membrane proteins here can be GFP It is good to have GFP tag localized inside the cell

8 Fluorescent proteins on the market

9 Excitation and emission

10 Multicolored fluorescent protein (neurones)

11 Transport among compartments Alberts et al. 2008

12 Also RNA can be differentially localized

13 RNA ZIP codes for localization Mikko Frilander

14 ZIP codes often motor protein bound Mikko Frilander

15 Delivering at the correct address 1. Localisation complex assembly starts already in the nucleus 2. Cytoplasmic mRNP is "matured“, nuclear export proteins removed, additional proteins attached. 3. mRNP is associated with motor and transport system and delivered to the destination 4. Delivered mRNP is anchored to the destination (for storage) or translated directly. Mikko Frilander

16 Localization of mRNA RNA hybridization in situ

17 classical technique, no alternative in developmental biology results often clear can be done without generating transgenic lines tedious only on “dead” samples

18 λN 22 system nuclear localization signal promoter viral RNA binding protein

19 Also mRNA can be differentially localized Ash1 mRNA localized to the tip of the daughter cell

20 Also mRNA can be differentially localized

21

22 Protein localization immunolocalization - fluorescently fluorescent dye attached primary antibodies secondary antibodies

23 Protein localization immunolocalization - immunogold electron microscope

24 Transport among compartments Alberts et al. 2008

25 Protein sorting – target peptides

26 Nuclear transport nucleoporins

27 Nuclear import

28

29 Mitochondrial transport TIM and TOM complexes decide at which side of mitochondria the protein will be transported.

30 Advanced confocal techniques FRAP photoactivatable FP FCS

31 FRAP region of interest Fluorescence Recovery After Photobleaching

32 FRAP

33 FRAP – bleaching curve

34 iFRAP inverse FRAP

35 iFRAP – dissociation of premRNA from specles

36 FRAP - advantages not only proteins (also other dyes)

37 your cells are moving high energy needed to bleach the ROI –can damage your material –long time needed to bleach usually only one ROI can be observed – time consuming FRAP – disadvantages

38 FRAP derivatives FLIP Fluorescence Loss After Photobleaching bleaching process is repeated during the experiment for studying general protein turnovers in compartments less often used continuous bleaching here

39 FRAP derivatives FLAP Fluorescence Localization after Photobleaching two fluorochromes on one protein– one bleached, non bleached as control

40 Intermezzo: story from a conference even top scientists can be wrong

41 Photoactivable proteins photoactivation (UV) non activated overlay

42 Photoactivable proteins Dronpa, Kaede, Eos – probably most popular

43 Photoactivable proteins Advantages: -most elegant, most convincing Disadvantages: -very weak signal -each material needs optimization

44 Remarks your material is 3D protein de novo synthesis in some experiments (e.g. cycloheximide stops translation)

45 FLIM Fluorescence Life Time Imaging Microscopy Fluorochromes excitation spectra emission spectra unique lifetime Lifetime sensitive to almost everything: pH ionic strength polarity other fluorochrome

46 FLIM - applications Protein-protein interactions (FRET-FLIM) (other lecture)

47 FLIM - applications lifetime decreased by site specific IgG injection phosphorylation assay

48 FCS Fluorescence Correlation Spectroscopy t + τ It is counted, how many times the fluorescent molecule comes through the focal plane. autocorrelation analysis Autocorrelation analysis: the way how to discriminate the diffusions speeds of particles.

49 FCS control membrane bound GFP and RFP (crosscorrelation curve) free GFP and RFP

50 FCS control membrane bound GFP and RFP (crosscorrelation curve) channel crosstalk threshold

51 FCS control membrane bound GFP and RFP (crosscorrelation curve) receptor with two labels channel crosstalk threshold

52 FCS receptor with two labels the crosscorelation curve is above threshold -> EGFR protein dimerizes Liu et al., 2007


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